The anesthetized rats were transcardially perfused with 0.9% saline. The eyes were excavated and the anterior segments were removed. The eyecups were fixed in 4% PFA for 1 h and dehydrated in graded sucrose solutions (20–30%) overnight at 4°C. The eyecups were then embedded in optimal cutting temperature compound (Tissue-Tek; Ted Pella, Inc., Redding, CA, USA) and snap-frozen at −80°C until they were sectioned (10 μm). The frozen sections were stored in a −20°C freezer. The slices were fixed in 4% PFA for 30 min and washed three times with PBS. They were then treated with 0.5% Triton X-100 (Beyotime, Shanghai, China) for 15 min. The slices were blocked with 10% goat serum in PBS for 1 h at room temperature and then incubated with a primary antibody overnight at 4°C. Antibodies directed against the following proteins were used: p-P38 (1:100, Cat #4511, CST, Boston, MA, USA), p-JNK (1:100, Cat #4668, CST, Boston, MA, USA), or p-ERK1/2 (1:100, Cat #4370, CST, Boston, MA, USA). The slices were then washed three times with PBS, incubated with the corresponding secondary antibody (1:1000, Cat #A21428, Invitrogen, Carlsbad, CA, USA) for 1 h, and counterstained with 4′,6-diamidino-2-phenylindole (diluted 1:1000; Sigma-Aldrich, St. Louis, MO, USA). Finally, the slices were scanned with a laser confocal microscope (Leica Microsystems, Bensheim, Germany).
Tissue tek
Manufactured by Ted Pella
Sourced in United States
Tissue-Tek is a laboratory equipment product designed for the processing and embedding of tissue samples. It provides a standardized and controlled environment for the preparation of tissue specimens for microscopic analysis. The core function of Tissue-Tek is to facilitate the infiltration of tissue samples with paraffin or other embedding media, ensuring the proper preservation and orientation of the samples prior to sectioning and staining.
Automatically generated - may contain errors
Lab products found in correlation
Laser confocal microscope, by Leica Microsystems (2 mentions)
Formaldehyde, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Ab49873, by Abcam (1 mentions)
Ab104139, by Abcam (1 mentions)
A28180, by Thermo Fisher Scientific (1 mentions)
Glass slide, by Thermo Fisher Scientific (1 mentions)
Leica cm3050 s cryostat, by Leica Microsystems (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Fluorescein, by Roche (1 mentions)
6 protocols using tissue tek
1
Immunofluorescence Analysis of Activated MAPK Signaling
2
Retinal Tissue Preparation for Western Blot
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Three days, 1 week, and 2 weeks after first laser treatment, the rats were anesthetized with intraperitoneal injection of 10% chloral hydrate and then were killed by cervical dislocation method. Both eyes were enucleated and postfixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 °C. After dehydration in graded sucrose solutions (20–30%), retinas were embedded in OCT compounds (Tissue-Tek; Ted Pella Inc, Redding, CA, USA) and stored at −80 °C. For western blot analyses, whole retinas and optic nerves were used immediately or stored at −80 °C until use.
3
Immunohistochemical Localization of SHP-1, pJNK, and GS
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The eyecups were fixed in 4% paraformaldehyde for 1 hour and dehydrated sequentially in 20% and 30% sucrose solutions at 4°C for 1 hour. After the anterior sections had been removed and the cups filled with optimal cutting temperature compound (Tissue‐Tek; Ted Pella), they were then frozen at −80°C, and cut in the sagittal direction (8‐μm‐ thick sections). After being blocked with 5% goat serum and permeated with 0.15% Triton X‐100 in PBS for 45 minutes, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti‐SHP‐1(sc‐7289, diluted 1:50; Santa Cruz Biotechnology); mouse anti‐pJNK (9255S, diluted 1:100; Cell Signaling Technology) and rabbit anti‐ glutamate synthase (GS) (ab49873, diluted 1:5000; Abcam). The sections were rinsed three times with PBS and incubated with a secondary antibody (A28180; Invitrogen; or 4414, Cell Signaling Technology) for 1 hour at room temperature. After the sections were rinsed three times, they were counterstained with Fluoroshield with DAPI mounting medium (ab104139; Abcam). The sections were then observed with a laser confocal microscope (Leica Microsystems).The immunofluorescence intensity in the images was measured with ImageJ software (National Institutes of Health).
4
Perfusion and Cryosectioning of Mouse Brain
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Preparation and staining of mouse brain sections were previously described in [56 (link)]. In brief, the mice were deeply anesthetized with 10 : 1 mixture of ketamine 50 mg/ml (Tekam) and xylazine 20 mg/ml (Seton) (dose 0.1 ml/10 gm i.p.) diluted in 1X phosphate-buffered saline (PBS, pH = 7.4) (1X PBS) and then briefly perfused intracardially (flow rate: 8-10 ml/min for 2-5 min) with 1X PBS. This was followed by 10 min of 4% paraformaldehyde freshly prepared (Sigma-Aldrich) or commercially available 4% formaldehyde (a dilution of 37% formaldehyde solution, Sigma-Aldrich, in 1X PBS); all solutions were adjusted to pH 7.4. To ensure complete tissue fixation, brains were removed carefully and postfixed into the same fixative for 1 h at 4°C and then cryopreserved in 20-30% sucrose/PBS at 4°C in preparation for sectioning. Brains were then embedded in OCT compound (Tissue-Tek®, Ted Pella, Inc.) and sectioned sagittally into 14-20 μm thick slices at -20°C using a Leica CM3050 S cryostat (Leica Microsystems). They were then mounted on glass slides (Fisher Scientific) and stored at -80°C for further use in cresyl and immunofluorescence studies. The second group of brains was gently dissected to isolate the frontal cortex according to the mouse brain atlas [57 ], and the samples were stored at -80°C for a Western blotting assay.
5
Fixation and Cryosectioning of Eyecups
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The eyes were enucleated, the cornea and lenses were removed, and the eyecups immersed in 4% paraformaldehyde for 30 minutes. Next, the eyecups were dehydrated in 10% sucrose, followed by 30% sucrose and embedded in OCT compounds (Tissue-Tek; Ted Pella Inc, Redding, CA, USA). The eye cups were frozen at −80 °C before cryostat sectioning.
6
Apoptotic Neuron Quantification in Retinal Transduction
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At 3 days after light exposure, the numbers of apoptotic neurons in blank-rLV-or OE-GILZ-rLV-transduced eyes were measured by TUNEL staining. The eyes were enucleated, and cornea and lenses were removed, and the eyecups were immersed in 4% paraformaldehyde for 2 hours. The eyecups were dehydrated in graded sucrose solutions (20%-30%) and embedded in OCT compound (Tissue-Tek; Ted Pella, Inc., Redding, CA, USA). The eyecups were then snap-frozen at À808C and sectioned (10 lm) 20 minutes later. Air-dried sections were then treated with reagents from the In Situ Cell Death Detection Kit and fluorescein (Roche). Briefly, sections were washed three times in phosphate-buffered saline for 10 minuters and incubated for 60 minutes in 0.1% Triton X-100. Sections were immersed in TUNEL reaction mixture (50 lL of enzyme solution added to 450 lL of label solution) at 378C for 60 minutes. After three rinses with 0.01 mol/L phosphatebuffered saline, the sections were counterstained with 4 0 ,6diamidino-2-phenylindole (Sigma-Aldrich Corp. St. Louis, MO, USA) and examined using microscopy (Leica Microsystems, Bensheim, Germany). In each section, two areas 500 lm from the optic nerve head were selected for imaging. In each visual field, the number of TUNEL-positive cells was counted and averaged. Only one section was chosen from each eye, and each group contained three eyes.
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